Many diseases are known to affect the human respiratory tract. For example, cystic fibrosis, which is the most common lethal inherited disease affecting the Caucasian population, has been linked to the production of a defective protein which appears to affect electrolyte transport across respiratory epithelial tissues. However, elucidation of the molecular basis of the cystic fibrosis transport defect has been hampered by the lack of suitable epithelial tissue available for study. Until now, the most readily obtainable cystic fibrosis epithelial tissue has been nasal mucosa acquired as a by-product of polypectomy or rhinoplasty. However, such specimens have been in very limited supply and often show reduced viability due to anaesthesia, trauma or infection. Moreover, increased use of intra-nasal steroid treatments in recent years has further reduced the availability of polypectomy tissue.
Researchers have consequently investigated other techniques for harvesting respiratory epithelial cells, such as nasal brushing or scraping. Nasal brushing involves the manual insertion of a cytology brush into the nasal passages of a living donor, such as a cystic fibrosis patient. Epithelial cells are collected by reciprocal and rotary movement of the brush on the donor's nasal mucosa. This technique is minimally-invasive and is well tolerated by adult and paediatric donors.
Previous attempts to culture brushed or scraped human nasal epithelial (BHNE) cells in vitro have not been successful. The primary difficulty facing tissue culturists has been the failure of BHNE cells to adhere to in vitro substrates for significant periods of time. Even when they do initially adhere, BHNE cells will usually peel off the chosen substrate after several days without flattening and replicating. Accordingly, such cells cannot be reliably used for biochemical and electrophysiological experiments or diagnostic testing, nor can they be amplified by serial subcultivation.
In an attempt to increase cell seeding efficiencies the applicant has investigated various protocols for enhancing BHNE cell adherence. For example, a wide variety of substrates have been investigated for culturing BHNE cells in vitro, including substrates coated with laminin, lectin, lipoteichoic acid, fibronectin and type I or type IV collagen. Using standard plating, washing and feeding procedures, most of the aforesaid substrates have demonstrated only minimal cell adherence.
The subjection of BHNE cells to a period of recovery after harvesting and prior to plating on the in vitro substrate also has no beneficial effect on BHNE cell seeding efficiencies. Other approaches for enhancing cell adherence have included treating the harvested cells with enzymes to break down the mucus and DNA gel typically entrapping the harvested BHNE cells and preventing cellular contact with the substrate surface. Another proposed protocol involves treating the BHNE cells with chemical activators in order to expose membrane adherence sites. However, such techniques have met with only limited success and often result in pharmacological side effects and abnormal cell morphology which renders the cultured BHNE cells unsuitable for scientific study.
Accordingly, the need has arisen for a readily reproducible method for culturing BHNE cells in vitro which has a high cell adherence and seeding efficiency.